1. Field of the Invention
This invention relates to a fluorescence measurement device for scanning a specimen.
2. Description of Related Art
In the epifluorescence mode of a fluorescence measurement the excitation and the emission beam path run in parallel for a certain distance and are separate from one another at a givenxe2x80x2 point. The separation is usually achieved by using a dichroic beamsplitter, which is capable of reflecting light below a given wavelength and transmits longer wavelength light. The necessity of having to use one such filter makes it difficult to rapidly change the excitation or the emission wavelength. In U.S. Pat. No. 5,751,417, describing a special type of confocal fluorescence microscope, the problem is circumvented by combining respectively separating excitation and the emission beam path without the aid of beam splitters, by employing dispersive elements, for example prisms. In doing so, the visual field is illuminated with a plurality of slit-shaped or point-shaped xe2x80x9clight windowsxe2x80x9d and the preparation is xe2x80x9cscannedxe2x80x9d by simultaneous movement of these illuminated fields. Published German Patent Application DE 43 30 347 A1 discloses a slit formed between two reflecting slit jaws, which allow to adjust the slit width, thus dividing the light beam, previously spectrally dispersed by a prism, into three spectral regions: one center wavelength region corresponding to the light passing the slit, one wavelength region above the center wavelength, being reflected by one jaw of the slit, and one wavelength region below the center wavelength, being reflected by the other jaw of the slit.
This invention is aimed at providing a fluorescence measurement device, which retains the indicated advantages of the device known from U.S. Pat. No. 5,751,417, but, by abandoning the need for parallel scanning and by focussing on individual xe2x80x9clight windowsxe2x80x9d, allowing a more easy implementation. Moreover, with such individual xe2x80x9clight windowsxe2x80x9d an improved stray-light suppression is achieved, and free, independent selection of the excitation wavelength, the emission wavelength and the emission bandwidth is allowed. In one special embodiment it is even possible to operate simultaneously with several excitation wavelengths and make accessible all the spectral information of the emission light for evaluation.
A primary objective of the invention is to provide a fluorescence-measuring device for scanning a specimen with a selection element.
This objective is achieved in accordance with the invention in that an excitation light is coupled to an excitation beam path by the selection element and guided to the specimen surface. Fluorescent light, which is emitted by the specimen, follows the excitation beam path in a reverse direction. A first dispersive element in the beam path causes a deviation of the emission beam from that of the excitation beam and allows the separation of the emission beam and the excitation beam on the selection element. The selection element includes a first area and a second area, thereby allowing selection between the excitation and the fluorescent light.
The boundaries of the second area are adjustable in order to allow the free selection of the wavelength range of the fluorescent light to be detected and to preset a desired Stoke""s shift. In this approach, the simple and flexible choice of the emission wavelength, emission bandwidth and the desired Stoke""s shift is advantageous.
In another embodiment of the present invention, it is advantageous to have several excitation and emission wavelengths measurements possible at the same time and confocality in two spatial directions enabled.
This invention separately provides for a special configuration of the selection element. This object is achieved in that the selection element preferably includes a first area and a second area. The first area and the second area capable of reflecting and are tilted against one another in order to reflect the excitation light and the fluorescent light in different directions.
In the following, embodiments of the invention are explained in detail by way of example using the attached drawings.